The phenotypic characterization of naturally occurring murine cytotoxic cells has been further developed to understand the origin, differentiation and normal function of this population. Cells from spleen, thymus, blood, bone marrow and liver have been characterized. Effector cell activity has been monitored against appropriate targets for both natural killer (NK) activity and natural cytotoxic (NC) activity. Phenotype has been determined by complement (C)-mediated, antibody-dependent cytotoxicity, by flow cytometry analysis (FCA) of immunofluorescence (IF) and by visual morphological assessment using Staphylococcus aureus (SpA) protein A dependent binding. Splenic and bone marrow subpopulations enriched for large granular lymphocytes (LGL) from nylon wool nonadherent cells subjected to density separation techniques have been characterized with a series of monoclonal antibodies (MoAb) to T-cell differentiation antigens, to myelomonocytic antigens and to other hematopoietic subsets. These studies have indicated a consistent failure to obtain a high level of enrichment and purity of LGL from spleen or bone marrow populations of untreated animals. This led us to examine cells from animals whose NK activity has been augmented by biological response modifiers (BRM). In addition, of LGL obtained from livers of BRM-treated mice have been compared to splenic subpopulations enriched for LGL. A pronounced heterogeneity in phenotype between the different populations has been observed. The phenotypic differences may be related to either the BRM used for NK augmentation or to the organ used as a source of cells. Characterization of bone marrow progenitors of NK activity has been initiated to determine whether the precursors share any of the markers found on mature, functional NK cells.